Method of modifying cellular differentiation and function and compositions therefor

ABSTRACT

A method and composition for enhancing biological response modifiers, such as promoting cellular differentiation, by using a Vitamin A or an analog thereof and sphingosine or an analog thereof.

The United States government has rights in this invention pursuant toGrant Nos. GM33369 and CA22294, National Institute of Health.

This application is a continuation of application Ser. No. 07/292,564,filed Dec. 27, 1988, now U.S. Pat. No. 5,170,876.

BACKGROUND OF THE INVENTION

The present invention relates to a method of effecting the modulation ofdifferentiation of cells by serum factors and inhibitors of ProteinKinase C. More particularly, the present invention relates to the use ofsphingosine and its analogs to promote cellular differentiation.

Differentiation is the modification of cells in structure or functionduring their course of development. Recent approaches to therapy forvarious types of cancer have focused on drugs that induce the maturationof the aberrant, differentiation-resistant cells causing the diseases.One example of such a compound is retinoic acid, which is a less toxicanalog of vitamin A. Retinoic acid has been found to be a potent inducerof differentiation in established myeloid cell lines, as well as primarycultures of cells isolated from patients with promyelocytic leukemia.

However, a problem exists in that certain cellular functions can becontrolled by biological response modifiers only under artificialconditions. It has been found that leukemic cells are less responsive inpatients than when isolated from bone marrow and cultured innutrient-rich medium supplemented with fetal calf serum. The success ofretinoic acid therapy for a leukemia patient is typically predicted bydetermining the ability of the compound to induce differentiation ofcells isolated from bone marrow and cultured in such medium. Clinicaltrials and case reports indicate that retinoic acid only inducesdifferentiation in some patients and that the effectiveness of retinoicacid therapy is limited because many patients still develop infectionsto which they ultimately succumb. Furthermore, in many of the patientswho do show improvement, the retinoic acid eventually ceases to causedifferentiation and the leukemic cells proliferate.

There exists a need, therefore, for an improved method of controllingcellular differentiation and function using biological responsemodifiers.

There also exists a need for such a method which encouragesdifferentiation under in-vivo conditions.

SUMMARY OF THE INVENTION

Protein Kinase C is an enzyme that participates in cellular responses tomany serum factors. According to the present invention, the addition ofan inhibitor of Protein Kinase C, such as sphingosine and its analogs,facilitates retinoic acid-induced differentiation. For example, it hasbeen found that treating cells with both retinoic acid and sphinganineproduce a more functional population of cells then those treated withretinoic acid alone.

The inhibitor can therefore be useful as an adjunct in the treatment ofdiseases in which the progression of the disease, or in which thetherapy for the disease, involves growth factors, hormones, or a varietyof drugs based on the mechanism of action of such compounds.

A specific example of the potential use of the present invention is theenhancement of leukemic cell differentiation in response to retinoicacid. The present invention may also be used in the treatment of cancer,arthritis, atherosclerosis, diabetes, inflammatory diseases, psoriasis,chronic granulomatous disease, and other diseases involving impairedfunction of cells in response to extra cellular stimuli.

It is an object of the present invention, therefore, to provide animproved method of controlling cellular differentiation and functionusing biological response modifiers.

It is also an object of the present invention to provide such a methodwhich encourages differentiation under in-vivo conditions.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 summarizes the uptake of [³ H] retinoic acid by HL-60 cells;

FIG. 2 summarizes the effect of sphinganine on retinoic acid-induceddifferentiation of HL-60 cells; and

FIG. 3 illustrates the respiratory burst of differentiated HL-60 cells.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to the use of an inhibitor of ProteinKinase C, such as sphingosine and its analogs, to enhance celldifferentiation in response to other agents.

The effects of different sera on RA-induced differentiation are shown inFIG. 1. The calls grown in serum-free medium show the most morphological(only 10% remained promyelocytic) as well as functional (82% ware NBT+)maturation after 4 days in RA. Serum has an inhibitory effect ondifferentiation by both criteria and varied with the source. FCS allowsthe most differentiation (25% remained promyelocytic and 58% were NBT+)while maturation in calf serum (HyClone, 43% were promyeloctyic and 43%NBT+), and human serum (44% were promyeloctic and 37% NBT+), was less.As is typically seen, a small percentage of the cells in each sera groupexhibit a more differentiated phenotype without addition of RA.

It has been found that different retinoic acid-induced differentiationcan be effected by sera. The effects of different sera on retinoicacid-induced differentiation of HL-60 cells are shown in Table I, below.HL-60 cells were originally isolated from a patient with acutemyelocytic leukemia and can be induced to differentiate into eithermonocyte/macrophage-like cells upon treatment with pharbol esters or1α,25-dihydroxyvitamine D₃ or granulocytes when placed in mediacontaining retinoic acid.

HL-60 cells (between passages 20 and 30) were adapted to growth in theRPMI 1640 medium supplemented with no serum, fetal calf serum, humanserum [type AB from male donors (Sigma)], or calf serum (all at 10%final concentration) and maintained by subculturing every 3 or 4 days ata density of 2.5×10⁵ cells/ml. Differentiation was assessed after 4 ofdays growth in either the presence or absence of 1 uM RA (added from a10 mM stock solution in DMSO to an initial cell number of 2.5×10⁵cells/ml) by morphology and nitro blue tetrazolium (NBT) reduction.Differentiation in response to retinoic and treatment was assessed bymorphology (the percentage of the cellular population in the variousstages of granulocytic differentiation) as well as functional assays(the activity of the NADPH oxidense as measured by the reduction ofnitro blue tetrazolium (NBT) when activated by phorbol esters.Morphology was judged on slides prepared with a Shandon Southerncytospin stained with Wright-Giemsa stain (Camco Quik II). The resultsfrom a representative determination are also shown in Table I.

These findings have been confirmed by repeating the measurements twicefor cells in serum-free medium and at least four times for those inserum-containing media. In each case, 200 cells were scored. Thepercentage of cells capable of reducing NBT was determined by countingthe number which contained precipitated formazan after a 30 minute, 37°C. incubation with an equal volume of NBT (1 mg/ml in 140 mM NaCl, 9.2mM Na₂ HPO₄, 1.3 mM NaH₂ PO₄, pH 7.4) and 160 nM phorbol 12-myristate13-acetate. The percentages shown represent the average of threedeterminations in which at least 200 cells were counted. In Table I,Blast=myeloblast, Pro=promyeloctye, Myelo=myelocyte,Meta=meta=myelocyte, Band=banded neutrophil, and Seg=segmentedneutrophil.

                  TABLE I                                                         ______________________________________                                        The results were as follows:                                                  Serum [RA]   Blast  Pro  Myelo  Meta Band Seq  NBT+                           ______________________________________                                        None  --     0      86   10      4   0    0    17%                                  1 uM   0      10   33     33   14   10   82%                            FCS   --     0      88    6      2   1    3     7%                                  1 uM   0      25   33     25   9    8    58%                            Calf  --     0      87   11      1   1    0     6%                                  1 uM   1      43   31     17   8    8    43%                            Human --     0      84   11      3   1    1     7%                                  1 uM   0      44   35     18   2    1    37%                            ______________________________________                                    

It was found, therefore, that cells grown in serum-free medium showedthe most morphological and functional maturation after 4 days inretinoic acid. Serum had an inhibitory effect on differentiation by bothcriteria, and differentiation varied with the source. Fetal calf serumallowed the most differentiation while maturation in calf serum andhuman serum was less.

It has been determined that the enzyme Protein Kinase C plays animportant role on the serum effects on retinoic acid-induceddifferentiation. It has also been found that sphinganine enhancesretinoic acid-induced differentiation. Sphinganine, or 2D- orD-erythro-2- or (2S,3R)-2 amino, 1.3-octadecanediol, is a constituent ofthe sphingolipids and is known to be a potent inhibitor of ProteinKinase C. To determine the effects of sphinganine on retinoicacid-induced differentiation of HL-60 cells, 5×105 cells plated in 2 mlof RPMI 1640 medium containing the indicated serum (10%) in 12-wellculture dishes (Costar) were treated with sphinganine (prepared as the1:1 molar complex with bovine serum albumin as previously described)alone or in combination with 1 uM RA. The sphinganine was added daily togive the following final concentrations: serum-free, 1.0 uM/day; FCS,2.5 uM/day; calf serum, 4.0 uM/day; human serum, 4.0 uM/day. The degreeof maturity of each group was assessed by morphology (open bars,expressed as the percentage of cells which had matured beyond thepromyelocyte stage) and NBT reduction (solid bars, determined asdescribed above). The results for the serum-containing groups, reportedas averages of at least 4 experiments shown ±S.E., are summarized inFIG. 2. In FIG. 2, C=control, RA=retinoic acid alone, and +Sa=retinoicacid plus sphinganine.

It was found that sphinganine had little effect on the retinoicacid-induced differentiation of HL-60 cells in serum-free medium;however, it increased the percentage of mature cells in allserum-containing media. The cells in fetal calf serum treated withretinoic acid and sphinganine differentiated to levels close to thoseseen with retinoic acid alone in serum-free medium (as judged bymorphology). Likewise, the percentage of mature cells after retinoicacid and sphinganine treatment in calf serum was similar to thoseobtained with retinoic acid in FCS. The percentage of NBT positive cellswas also increased although to a lesser extent. Treatment withsphinganine alone (i.e., without retinoic acid) also slightly increasedthe percentage of differentiated cells.

It has also been found that the initial rate of superoxide production inresponse to the chemotactic peptideN-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP) can be increasedwith retinoic acid alone relative to indifferentiated cells. It wasfurther found that the rate of fMLP-initiated respiratory burst wasfurther increased by treatment with both retinoic acid and sphinganine.

During granulocytic maturation, cells normally acquire the ability toproduce superoxide when activated by physiological stimuli such as thechemotactic peptide N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP)and phorbol 12-myristate 13-acetate (PMA). Whereas phorbolester-stimulated superoxide production requires only a functional NADPHoxidase, the response to fMLP entails the presence of the fMLP receptorand the coupling mechanisms to the oxidase. HL-60 cells induced todifferentiate by dimethylsulfoxide and dimethylformamide appear todevelop all these components and produce superoxide when stimulated byfMLP, as reported by Skubitz et al., Blood 59,586 (1982) and Brandt etal. Cancer Res. 41,4947 (1981). However, cells treated with retinoicacid show little activation in response to this peptide, suggesting thatmaturation induced by this agent is incomplete. To determine whethersphinganine treatment affects the ability of retinoicacid-differentiated HL-60 cells to be activated by fMLP, the rate ofsuperoxide production in response to both this peptide and PMA wasquantitated.

Cells were grown in RPMI 1640 medium supplemented with 10% calf serumwith no additions (control), 1 uM retinoic acid, 4.0 uM sphinganine (Sa,added on days 1 and 2 only), or both 1 uM retinoic acid and 4.0 uMsphinganine (added on days 1 and 2). After 4 days, the cells werecollected by centrifugation and washed with phosphate buffered saline(PBS, 0.1 g/l CaCl₂, 0.2 g/l KCl, 0.2 g/l KH2PO₄, 0.1 g/l MgCl₂.6H₂ O,8.0 g/l NaCl, and 2.16 g/l Na₂ HPO₄.7H₂ O) containing glucose (1 g/l).The cells were then resuspended in PBS+glucose at a density of 1×10⁷cells/ml. The respiratory burst of each group was quantitated bymeasuring the reduction of cytochrome c (25 mg/ml) as reflected by thechange in absorbance at 549 nm minus 540 nm measured with a SLM AmincoDW-2000 spectrophotometer in the dual wavelength mode. Each measurementwas done with 2.4×10⁶ cells in a total volume of 2.4 ml. The respiratoryburst shown in the top panel of FIG. 3 was initiated by fMLP (1 uM)while that in the lower panel was initiated with PMA (100 nM).

As summarized in FIG. 3, the response to fMLP in cells treated withretinoic acid alone increased by 220% relative to the undifferentiatedcells, and the rate of respiratory burst was increased by 410% in HL-60cells treated with both retinoic acid and sphinganine relative to thecontrol. Some variability in the fMLP-stimulated rate of superoxideproduction was seen between different groups of cells: averages ofresults from 5 separate experiments show that this rate increased 75±30%for retinoic acid alone and299±100% for retinoic acid plus sphinganine.

The rate of superoxide production in response to PMA was increased incells differentiated with retinoic acid by 210% and with retinoic acidplus sphinganine by 230% both relative to the control rate in amountsconsistent with the increases in NBT reduction above. The averageincrease, calculated from five experiments, was 82±24% for retinoic acidtreated cells and 123±60% for cells treated with both retinoic acid andsphinganine. Therefore, because the increase in superoxide productionwhen retinoic acid and sphinganine are used together is greater whenstimulated by fMLP than by PMA, it appears that the long-chain base aidsthe HL-60 cells to differentiate more completely so that they becomeresponsive to physiological agonists.

It has been found, therefore, that serum influences the ability ofretinoic acid to induce cellular differentiation. Also, the addition ofsphinganine has been found to not only increase the percentage ofcellular population when differentiated, but also promotes morefunctional maturation of the cells. The use of sphingosine and itsanalogs as enhances of biological response modifiers provides means forovercoming the frequent failures of retinoic acid therapy, and providesdifferentiation under in-vivo conditions.

Furthermore, the present invention is not limited to retinoic acid, butapplies to all systems with key processes that are inhibited bysphingosine and its analog and are involved in a biological process ofinterest.

For example, a daily intervenous dosage of approximately 50 mg and uptto 0.5 mg may be used, which compares to clinical studies of13-cis-retinoic acid where doses of 50 to 100 mg per day are used.

What is claimed is:
 1. A method of promoting cellular differentiationand maturity of myelocytic and promyelocytic cells capable ofdifferentiation, as assessed by morphology or nitro blue tetrazoliumreduction, comprising the step of treating said cells with an effectiveamount of sphingosine or an analog thereof.
 2. The method of claim 1,wherein the analog of sphingosine is sphinganine.
 3. A method ofincreasing superoxide production by granulocytes that are responsive toVitamin A or an analog thereof, comprising the step of treating saidgranulocytes with effective amounts of Vitamin A or an analog thereofand sphingosine or an analog thereof.
 4. The method of claim 3, whereinsaid analog of Vitamin A is retinoic acid.
 5. The method of claim 3,wherein said analog of sphingosine is sphinganine.
 6. The method ofclaim 3, wherein said superoxide production is in response to achemotactic peptide.
 7. The method of claim 6, wherein said chemotacticpeptide is N-formyl-L-methionyl-L-leucyl-L-phenylalanine.